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Abstract

Alginate is a family of industrially important polysaccharides composed of irregular sequences of 1-4 linked β-D-mannuronic acid (M) and α-L-guluronic acid (G). They are widely used industrially as iscosifiers and gelling agents. Medical applications include utilization as dental impression materials, wound dressings and as an encapsulation matrix for cell transplants in the treatment of various diseases. Some alginates are immunogenic or have anti-tumor activity.

Commercial alginates are extracted from brown seaweeds, but the polymer is also produced by members of the bacterial genera Pseudomonas and Azotobacter. Probably in all species the alginate is first synthesized as polymannuronic acid, and then the guluronic acid moieties are introduced at the postpolymerization level by the action of mannuronan C-5- epimerases. Azotobacter vinelandii encodes a family of 7 secreted, Ca2+ -dependent mannuronan C-5-epimerases, AlgE1-7, which are composed of varying numbers of two types of structural modules, A and R. The A-modules alone are catalytically active, but their reaction rates are substantially increased when linked to at least one R-module. The various epimerases introduce different sequential M/G patterns into the alginate. AlgE4 strictly forms alternating sequences (MG-blocks), while AlgE2, AlgE5 and AlgE6 produce blocks of contiguous G-residues (G-blocks) which vary in length and distribution. AlgE1 and AlgE3 contain two catalytic modules, one G-block forming module and one strictly MG-forming module. AlgE7 is special in that it is both an epimerase and an alginate lyase.

A. vinelandii produces alginate both as a vegetative capsule and as an essential constituent of the coat surrounding a resting stage designated cyst. The AlgE-epimerases are believed to play an important role in designing the composition and properties of the cyst alginate, and they are secreted at different stages during cyst formation and germination.

In this study the structure-function relationship of the mannuronan C-5-epimerases AlgE4 and AlgE2 has been studied. The 377 amino acids long AlgE4 A-module has been expressed recombinantly in Escherichia coli, and after purification the structure of the protein was solved by X-ray crystallography by the group of Dr. B. Dijkstra at the University of Groningen, Groningen, The Netherlands. The protein was found to fold into a right-handed β-helix with 4 parallel sheets, PB1, PB2a, PB2b and PB3. Like in other enzymes with this fold, the catalytic site seems to be positioned in a groove, formed by PB1 and the turns T1 and T3. The catalysis is believed to involve the amino acids Tyr149, Asp152 and His154, but other amino acids in their vicinity are also required for activity. The predicted substrate groove is highly basic in ontrast to the rest of the protein, which is generally acidic, accommodating binding of the acidic alginate polymer.

By interchanging internal corresponding parts of the A-modules of AlgE2 and AlgE4, a series of hybrid enzymes have been constructed. All these hybrids were active and NMR spectroscopical analyses showed that several formed M/G patterns differing from and intermediating those of the parent enzymes. Amino acids in the central part of the sequence were shown to highly influence the epimerization pattern and they correspond to a band of amino acids at the C-terminal proximity of the proposed catalytic site. The same amino acids seem to influence the level of processivity of the enzymes and the reaction rate.

For all previously studied mannuronan C-5-epimerases it has been shown that they act according to a non-random mode of action. Interestingly, one of the hybrid enzymes, designated KA1, seemed to generate alginates as if it had acted in a near-random mode. The reaction rate of the enzyme was however low, implying that it might bind poorly to the substrate.

A new epimerase gene from Pseudomonas syringae pv glycinea PG4180, which partially showed high sequence similarity with the A. vinelandii algE-epimerases, was characterized and found to encode a bifunctional enzyme. The enzyme, designated PsmE, contained two catalytic sites comprising mannuronan C-5- epimerase and mannuronan O-acetyl hydrolase activity, respectively. The deduced enzyme is composed of 1610 amino acids, and showed a complex modular structure, A-R1-R2-M-R3-N-RTX-S. The A- and Rmodules are similar to those present in the A. vinelandii AlgE-epimerases, and the A-module was found to be 53-61% identical to the AlgE-A-modules. The Mmodule is similar to the Ca2+ -binding dystroglycan-type cadherin-like domains, RTX is similar to the R-modules and the Ca2+-binding modules of RTX-toxins, while the N-module comprises the acetyl hydrolase activity. Bacterial alginates are partly acetylated, and such modified residues cannot be epimerized. It was therefore very interesting to note that the acetyl hydrolase activity of PsmE enabled the enzyme to epimerize such alginates to a much greater extent than the AlgE epimerases.

PsmE was found to form some of the longest G-blocks known, and a hybrid (PsmeA4R) between its catalytic A-module and the R-module from AlgE4 was able to transform an alternating alginate into a 96% G-alginate. The enzyme was active on various types of alginates, and when acting on pure mannuronan, the epimerase activity was highest around 37°C in a 50 mM Mops buffer at pH 6.8, containing 0.8 mM CaCl2. At higher temperatures the enzyme was rapidly inactivated.

Initial studies using a promoter-probe vector to investigate the putative algE-epimerase promoters, showed that the genes are regulated independently of each other and that they all contained promoter regions. The algE4-promoter was found to be active during the encystment process, while the promoters of algE2, algE3 and algE5 were transcribed in the vegetative state. The algE1 promoter was not active in the vegetative stage, but it was activated in the encystment process and possessed the highest activity found. The promoters of algE6 and algE7 were mainly transcribed during germination.

A biological study of an AlgE7 knock-out mutant revealed that the enzyme, which possesses a lyase activity, is not required for cyst germination. Work has also been initiated to knock out the other epimerase genes in A.vinelandii.